Canine coronavirus vaccine from feline enteric coronavirus

ABSTRACT

This invention provides a vaccine which contains, per dose, an effective immunizing amount of an inactivated feline enteric coronavirus and a suitable carrier. The vaccine of this invention may also contain an adjuvant, an effective immunizing amount of a second inactivated virus and a an effective immunizing amount of an inactivated bacteria. Additionally provided by this invention is a method of immunizing a dog against disease caused by canine coronavirus involving administering to the dog a dose of the vaccine of this invention. The method of this invention may also involve administering one or more additional doses of vaccine to the dog, immunizing the dog against disease caused by a second virus and immunizing the dog against disease caused by a bacteria.

This application is a continuation-in-part of U.S. Ser. No. 08/024,165,filed Feb. 26, 1993 now abandoned. Throughout this application variouspublications are referenced by arabic numerals within parentheses. Fullcitations for these publications may be found at the end of thespecification immediately preceding the claims. The disclosure of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

BACKGROUND OF THE INVENTION

Coronaviruses belong to the family Coronaviridae and are composed of asingle genus of large, enveloped viruses which infect a variety ofmammalian and avian species. The virus is heat labile but acid stablewhich contributes to the ability to survive passage through the stomach.Virions of most coronaviruses contain three proteins; the phosphorylatednucleocapsid protein, N; a small membrane embedded glycoprotein, M; anda large petal-shaped peplomer glycoprotein, S. The M protein issynthesized on ribosomes bound to the rough endoplasmic reticulum andaccumulates in the Golgi apparatus and is believed to determine the siteof virus budding from the infected cell. The S protein mediates many ofthe biological properties of the virus, such as attachment to cellreceptors, penetration, cell-fusion, and is the major target forvirus-neutralizing antibodies. A proportion of the S glycoprotein whichis not incorporated into virions is transporter to the plasma membraneof the cell where it remains bound to the surface of the cell.

Canine coronavirus (CCV) was first reported by Binn et al. (1) Theisolate was recovered from dogs suffering from diarrhea during anepizootic in Germany. Keenan et al. (2) reported that the isolated viruswas capable of inducing intestinal lesions in susceptible neonatalpuppies.

CCV gastroenteritis in dogs is highly contagious, and followingexposure, the virus rapidly spreads throughout the small intestine. Theincubation period is generally 24 to 48 hours but has been as long asone to four days, as reported by Keenan et al. (2) Following oralexposure, the virus enters and colonizes in the upper duodenum. Viralreplication results and the infection then proceeds caudally throughoutthe entire small intestine within days. As a result of the enteritis,the CCV may also spread locally to the regional mesenteric lymph nodes.

The replication of the CCV is localized within the digestive andabsorptive cells that are located in the upper two-thirds of theintestinal villi. The viral replication causes death and desquamation ofthe intestinal epithelial cells resulting in shortening of theintestinal villi. The desquamating cells containing infectious virus area source of infection for more caudal segments of the intestines and thefeces. Loss of digestive enzymes and absorptive capacity results indiarrhea, and dehydration of puppies. Deaths have occurred within aslittle as 24 to 36 hours after onset of clinical signs despite goodsupportive care.

Most dogs are afebrile and pathological changes are usually notdetected. Puppies are most severely affected and the most common causeof deaths in neonates is from severe dehydration. Severity of thedisease can be enhanced when in combination with other enteric caninepathogens (3). Stress, breed and environmental conditions are additionalfactors that affect the severity of the disease. Animals exposed to CCVdevelop virus neutralizing (vn) antibody that is detectable in theserum. However, CCV infection is not systemic. Therefore, serum (SVN)antibody titers are indicative of exposure but not of protection. Thereis no effective antiviral treatment for CCV enteritis. Treatment issupportive with fluid replacement and electrolyte therapy to controlemesis and diarrhea and to prevent concurrent infections.

Mucosal immunity appears to play an important role in the protectionagainst CCV enteritis. Appel et al., (4) reported that local immunity,possibly mediated by IgA antibodies in the intestine, was responsiblefor protection against CCV infection, in a manner similar totransmissible gastroenteritis virus (TGEV) of pigs.

Since 1983, two types of vaccine have been used in an attempt to controldisease conditions associated with CCV infections. (5,6) A modified livevaccine introduced in 1983 by Fort Dodge Laboratories, Iowa, intended toprotect dogs against CCV was soon demonstrated unsafe and subsequentlywithdrawn due to adverse reactions. In 1985, an inactivated vaccine toprotect dogs against CCV was introduced by Fort Dodge Laboratories andhas been shown to be safe and efficacious. Later, another inactivatedCCV product was introduced by Smith Kline Beecham to protect dogsagainst CCV enteritis.

In 1978, the antigenic similarities of CCV, TGEV, feline infectiousperitonitis virus (FIPV) and human coronavirus 229 E were reported.(7,8) The close antigenic properties shared by these viruses allowed forthe development of serologic assays and experimental vaccines. Anotherfeline coronavirus, feline enteric coronavirus (FECV), has recently beenshown to be antigenically related to this group. The dominant antigenicdeterminants of the S protein are cross reactive between these virusesand afford the opportunity for use as vaccines in the distantly relatedanimal species (9). However, some cross-protection studies with theseviruses have offered little protection in the heterologous species (1,10, 11).

The present invention is directed to a new vaccine composition forprotection against CCV. It provides a method of preventing CCV infectionin dogs by administering a vaccine prepared from FECV. Theimmunodominant regions of the S protein of FECV are antiqenicallysimilar to corresponding components on the CCV virion. The S protein ofFECV provides a method of preventing CCV in dogs by priming theimmunocompetent cells on the mucosal surfaces of the intestine,providing an immunological memory mechanism for protection against CCVinfection. In addition, sufficient heterogenicity between the S proteinsof FECV and CCV provides an opportunity for the FECV vaccine to overcomelevels of CCV maternal antibodies which might neutralize homologous CCVvaccines.

SUMMARY OF THE INVENTION

This invention provides a feline enteric coronavirus vaccine whichcomprises per dose an effective immunizing amount of a modified felineenteric coronavirus and a suitable carrier. In the presently preferredembodiment of this inventions the modified feline enteric coronavirus isan inactivated feline enteric coronavirus. The vaccine provided by thisinvention may further comprise an adjuvant in an amount effective toenhance the inmmunogenicity of the modified virus. This invention alsoprovides a vaccine comprising a modified feline enteric coronavirus anda second modified virus. This invention further provides a vaccinecomprising a modified feline enteric coronavirus and a modifiedbacteria.

This invention provides a method of immunizing a dog against diseasecaused by canine coronavirus which comprises administering to the dog adose of the vaccine of this invention. Also provided is the method ofthis invention further comprising administering to the dog one or moreadditional doses of the vaccine at a suitable interval of time afteradministration of the preceding dose. The method of this invention maycomprise immunizing a dog against disease caused by canine coronavirusand disease caused by a second virus. The method of this invention mayalso comprise immunizing a dog against disease caused by caninecoronavirus and disease caused by a bacteria.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1: Summary of Mucosal IgA antibody to CCV S-protein levels in fecalsamples.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a feline enteric coronavirus vaccine whichcomprises per dose an effective immunizing amount of a modified felineenteric coronavirus and a suitable carrier.

For the purposes of this invention, an "effective immunizing amount" ofa modified feline enteric coronavirus is any amount of the modifiedvirus effective to immunize a vaccinated animal against disease causedby virulent virus. Typically, the effective immunizing amount of themodified feline enteric coronavirus is an amount which contains greaterthan about 5 micrograms of S viral antigen. Desirably, the effectiveimmunizing amount of the modified feline enteric coronavirus is anamount which contains from about 5 micrograms to about 7.5 micrograms ofthe S viral antigen. Example 1D, infra, provides a method forcalculating the S viral antigen content of the vaccine composition.

For the purposes of this invention, a "modified" feline entericcoronavirus may be a modified live virus, an inactivated virus, arecombinant virus or a viral subunit. A "modified live" virus is avirulent or attenuated virus capable of eliciting an immune responsefrom a vaccinated animal. As used herein, an "inactivated" virus is avirus which is not capable of infecting an animal's cells, but which canstill elicit an immune response from an animal vaccinated with thevirus. A "recombinant" virus is a genetically engineered immunogenicvirus which is incapable of infecting an animal's cells. The viralsubunit vaccine of this invention may comprise the feline entericcoronavirus S glycoprotein in an antigenic form. Thus, this inventioncontemplates a vaccine comprising an intact virus or an antigenic viralsubunit. The effective immunizing amount of the intact virus is thenumber of viral particles containing an amount greater than about 5micrograms of the S viral protein. The effective immunizing amount ofthe subunit vaccine is an amount greater than about 5 micrograms ofviral S glycoprotein.

In the presently preferred embodiment of this invention, the modifiedfeline enteric coronavirus is an inactivated feline enteric coronavirus.Feline enteric coronavirus may be inactivated by contacting the virus,as described hereinbelow, with an agent selected from the groupconsisting of binary ethylenimine, formalin or β-propriolactone. In thepresently preferred embodiment of this invention, the feline entericcoronavirus is inactivated by contacting the virus with binaryethylenimine.

"Suitable carriers" for use with a modified feline enteric coronavirusin a vaccine in accordance with the practice of this invention aresolutions suitable for suspending modified live viruses andadministering to animals. Such suitable carriers are well known to thoseskilled in the art and include, but are not limited to, aqueous buffers,e.g., phosphate buffers.

The vaccine of this invention may further comprise an adjuvant in anamount effective to enhance the immunogenicity of the modified felineenteric coronavirus. For the purposes of this invention, an "adjuvant"is a composition of matter which enhances the response of an animal'simmune system to a viral antigen when the antigen is administered withthe adjuvant. The vaccine of this invention may comprise an adjuvantselected from the group consisting of aluminum hydroxide, saponin oraluminum phosphate. In the presently preferred embodiment of thisinvention, the adjuvant is aluminum hydroxide.

For the purposes of this invention, an "effective amount" of an adjuvantis any amount of the adjuvant effective which, when it is administeredwith the antigen to an animal, is effective to enhance the animal'simmune response to the antigen. Typically, the effective amount of theadjuvant is from about 3% by volume of the dose of vaccine to about 10%by volume of the dose. Desirably, the amount of the vaccine is an amountabout 5% by volume of the dose.

Also provided is the vaccine of this invention further comprising aneffective immunizing amount of a second modified virus. The secondmodified virus may be a modified live virus, an inactivated virus, anattenuated virus or a viral subunit, as described hereinabove. Thevaccine of this invention may comprise a second modified virus selectedfrom the group consisting of a modified canine distemper virus, amodified canine adenovirus, modified canine parvovirus, modified canineparainfluenza virus or a modified canine herpesvirus.

The vaccine of this invention may further comprise an effectiveimmunizing amount of a modified bacteria, e.g., a modified Leptospira.As used herein, a "modified" bacteria is an intact bacteria capable ofeliciting an immune response in an animal to which the modified bacteriais administered, but which is incapable of causing disease in theanimal. The modified bacteria may be a modified live bacteria, aninactivated bacteria, or a bacterial subunit, i.e., an antigenic proteinderived from the bacteria. For the purposes of this invention, an"effective immunizing amount" of a modified bacteria is any amount ofthe modified bacteria effective to elicit an immune response in ananimal to which the modified bacteria is administered. Effectiveimmunizing amounts of modified bacteria are well known to those skilledin the art or may be readily determined by them without undueexperimentation. Typically, an effective immunizing amount of a modifiedlive or inactivated bacteria is an amount greater than about 10⁷cells/ml, desirably an amount from about 10⁷ cells/ml to about 10¹²cells/ml. Typically, an effective immunizing amount of a bacterialsubunit is an amount greater than about 10 micrograms of the bacterialsubunit, desirably, an amount from about 10 micrograms of the subunit toabout 1,000 micrograms.

This invention provides a method of immunizing a dog against diseasecaused by canine coronavirus which comprises administering to the dog adose of the vaccine of this invention. Typically, the dog is at leastabout six weeks old when the vaccine is administered. Desirably, the dogis from about six weeks old to about nine weeks old when the vaccine isadministered. The vaccine of this invention may be administered to thedog by any means generally accepted by those skilled in the art ofadministering vaccines to dogs. Examples of such means of administeringvaccines include, but are not limited to, subcutaneous injection orintramuscular injection.

The method of this invention may further comprise administering to thedog one or more additional doses of the vaccine, each additional dose ofvaccine being administered at a suitable interval of time afteradministration of the preceding dose. The method provided by thisinvention thus contemplates administering multiple doses of the felineenteric coronavirus vaccine to a dog. Administration of more than onedose of a vaccine is intended to enhance the immune response of thevaccinated animal to viral antigen. Administration of each dose shouldbe separated by an interval of time suitable to allow for an enhancedimmune response. A "suitable" interval of time between administration ofmultiple doses of a vaccine to an animal is therefore any interval oftime between administration of doses of a vaccine sufficient to causethe animal to generate a greater immune response than administered asingle dose. Typically, the suitable interval of time betweenadministrations of multiple doses of the feline enteric coronavirusvaccine of this invention is from about two weeks to about five weeksDesirably, the interval of time is from about two weeks to about threeweeks.

This invention contemplates administration of a booster dose of themodified feline enteric coronavirus vaccine. Preferably, the boosterdose is administered approximately one year after the previousadministration of vaccine. Administration of a booster dose of a vaccineto a dog is a generally accepted veterinary practice, designed to insurethat the dog remains capable of generating an immune response to viralinfection throughout its life.

Also provided is a method of immunizing a dog against disease caused bycanine coronavirus and disease caused by a second virus which comprisesadministering to the dog the vaccine of this invention, wherein thevaccine comprises the modified feline enteric coronavirus of thisinvention and a second modified virus. The second modified virus may beselected from the group consisting of a modified canine distemper virus,a modified canine adenovirus, a modified canine parvovirus, a modifiedcanine parainfluenza virus or a modified canine herpesvirus.

This invention provides a method of immunizing a dog against diseasecaused by canine coronavirus and disease caused by a bacteria whichcomprises administering to the dog a dose of the vaccine of thisinvention, wherein the vaccine comprises an effective immunizing amountof a modified feline enteric coronavirus and an effective immunizingamount of a modified bacteria. The modified bacteria may, but is notrequired to be, a modified Leptospira.

This invention will be better understood from the Examples which follow.However, those skilled in the art will readily appreciate that thespecific examples detailed are only illustrative of the invention asdescribed more fully in the claims which follow thereafter.

EXAMPLES Example 1

Vaccine Preparation

As discussed previously, the vaccine of this invention is produced usinga modified feline enteric coronavirus which can be a modified livefeline enteric coronavirus, an inactivated feline enteric coronavirus, arecombinant feline enteric coronavirus or a viral subunit of a felineenteric coronavirus, each being effective to protect an animal againstcanine coronavirus. In this example, the preparation of the vaccineusing an inactivated feline enteric coronavirus is discussed.

A. Preparation of Cell Cultures

Crandall feline kidney (CRFK) cells, available from the American TypeCulture Collection, ATCC No. CCL 94, or a cloned population identifiedas FK-CU are expanded as monolayers in plastic cell culture vessels inDulbecco's Minimal Essential Medium (DMEM) supplemented with vitamins,essential amino acids, 2 mg/ml sodium bicarbonate and 0.584 mg/mlL-glutamine Virology, Vol. 8: p. 396 (1959) and Virology, Vol. 12: p.185 (1960)!. As a preservative, 30 μg/ml gentamicin is added. Cells aregrown in culture vessels to 100% confluency, approximately 2×10⁵ cellsper cm² of growth surface area, using standard cell culture techniques.Generally, plastic roller bottles with a growth surface of 850 cm²,containing 200 ml of DMEM supplemented with 5% fetal bovine serum, and1.5×10⁸ cells are used for vaccine production. The cell cultures areinitiated by seeding approximately 1.5×10⁷ cells into 200 ml of growthmedium in a roller bottle on a roller bottle rotator at 0.25 to 2 rpm at35° C. to 38° C.

B. Inoculation of Cell Cultures with Feline Enteric Coronavirus andHarvest of Cultures

Isolates of feline enteric coronavirus, such as the WSU 79-1683 strain,available from the American Type Culture Collection, ATCC No. VR-989, orother suitable feline enteric coronaviruses may be used formanufacturing an inactivated CCV vaccine. Feline enteric coronavirus isreceived from the ATCC at the ninth cell culture passage is adapted togrow in the feline kidney cell line by two serial passages. The MasterSeed Virus MSV X is, therefore, the 11th passage. To establish purity,the Master Seed Virus is tested in accordance with 9 C.F.R. §§ 113.27,113.28 and 113.55. The passage level of the vaccine is no more than fivepassages from the Master Seed Virus.

CRFK stock cells are stored at -70° C. or lower. Virus Master Seed isstored at -70° C. Working seed and production seed viruses are stored at-40° C.

For inoculation of the roller bottles with virus, the growth medium isdecanted from the monolayers which are 95% to 100% confluent. The mediumis replaced with 100 ml of DMEM, without bovine serum, containingsufficient seed virus to achieve a minimum multiplicity of infection(MOI) of 0.01. An 850 cm² roller bottle contains 1×10⁸ to 4×10⁸ cells.At a MOI of 0.01, 2×10⁸ times 0.01 yields 2×10⁶, which is divided by theantilog of the seed titer. Virus seed and product harvest yield 10⁵.0 to10⁸.0 TCID₅₀ per ml. Thus, 2×10⁶ divided by 1×10⁶ (antilog of 10⁶.0)yields 2 ml of the seed used to add to the 98 ml of maintenance mediumcovering the cells in the 850 cm² roller bottle. For 40 such rollerbottles, 4000 ml of DMEM containing 80 ml of virus seed would be used asthe inoculum.

After decanting the growth medium from the rollers, 100 ml of thediluted virus would be added to each roller bottle. Inoculated culturesare incubated at 35° C. to 38° C. Infection is apparent by the typicalcytopathic effect (CPE) on the cell monolayer. The CPE consists ofpatches of rounded cells. Prior to harvest, cultures can be inspectedmicroscopically and macroscopically and only material that shows noindication of contamination is harvested. The fluids containing virusalong with the cellular material are harvested at 48 to 72 hourspost-infection without further purification, dispensed in containers andstored at -50° C. or below. For this example, 4000 ml of viral fluidswould be stored frozen.

C. Inactivation of Virus

For inactivation, the viral fluids are thawed in a 37° C. water bath,combined in a common container and inactivated by binary ethylenimine(BEI). The BEI solution is prepared by adding 2.05 g of2-bromoethylamine hydrobromide and 0.8 g of sodium hydroxide to 100 mlof water and incubating in a 37° C. water bath for one hour to cyclizeand form the BEI. The BEI solution is added to the viral fluids to afinal concentration of 1%, by volume. For this example, the 4000 ml offrozen viral fluids would be thawed and warmed to 37° C. and 40 ml ofthe BEI is added to the viral fluids. The viral fluids and BEI are mixedthoroughly and incubated at 37° C. for 24 to 48 hours. To confirmcomplete viral inactivation, two ml of the inactivated viral fluids aretested for the absence of residual live virus by inoculation onto amonolayer of feline cells. The inoculated cells are incubated at 37°C.±2° C. for seven days, then subpassaged. The subpassaged cells arestained with a fluorescein labeled polyclonal or monoclonal antiserum toFECV and examined under an ultraviolet microscope for absence of FECVfluorescence. At the end of the inactivation period, the BEI isneutralized by adding a cold (4° C.±2° C.) sterile 1 molar solution ofsodium thiosulfate to the reaction mixture to give a final concentrationof 0.25%, by weight. For this example, 10.1 ml of the 1 molar solutionof sodium thiosulfate is added to the reaction mixture.

D. Quantitation of the Vaccine

An enzyme linked immunoassay (ELISA) technique was developed accordingto standard methods and used to calculate the effective immunizingamount of the FECV vaccine. Monoclonal antibodies specific for thepeplomer (S) antigen of canine coronavirus and polyclonal antibodiesagainst CCV were prepared according to known methods. (12, 13) ELISAplate wells coated with a monoclonal antibody specific for the peplomer(S) antigen of canine coronavirus is reacted with diluted FECV testsamples and subsequently treated with a blocking buffer containingnonfat dry milk. After washing, diluted polyclonal antibody against CCVis added to the wells, the wells were washed again and then goatanti-cat IgG horse radish peroxidase (available from Kirkegaard andPerry Laboratories, Inc. Gaithersburg, Md.) is added. After a thirdwashing, 2,2'-azido-di- 3-ethylbenzthiazoline sulfonate (6)! is added tothe wells, and the reaction is stopped by addition of sodium dodecylsulfate. Absorbance of the color reaction in optical units is read at405 nm with an ELISA reader. The concentration of the FECV antigen inthe test is calculated from a standard curve generated by simultaneoustesting of dilutions of a known concentration of FECV. The inactivatedFECV vaccine of the invention is standardized by this ELISA technique tocontain an S antigen content of at least 5 micrograms.

E. Addition of Adjuvant and Preservative

This standardized vaccine preparation is then adjuvanted by mixing theinactivated viral fluids with aluminum hydroxide, to give a finaladjuvant concentration of 5%, by volume. For this example, thestandardized product would require the addition of 34,000 ml ofphysiological saline to the 4000 ml of inactivated FECV fluids for an Santigen content of 5 micrograms. This total volume then would be 38,000ml and to this, 2000 ml of aluminum hydroxide would be added for a finalconcentration of 5% by volume. As a preservative, merthiolate is addedto the adjuvanted inactivated vaccine to a final concentration of1:10,000 by volume.

Example 2

Interference Study

To demonstrate that the canine coronavirus vaccine containing felineenteric coronavirus does not interfere with the antibody response toother canine antigens, puppies were vaccinated with the CCV vaccine incombination with a vaccine containing modified live virus andinactivated leptospira antigens. All vaccines were formulated at theminimal release dose.

                  TABLE 1                                                         ______________________________________                                        Canine Coronavirus Vaccine Interference Study                                 Antibody response.sup.a  (GMT) post 2nd vaccination.sup.c :                                                           Leptospira                            Vaccine                                 Cani-                                 Group.sup.d                                                                             CDV    CAV.sub.2                                                                             CPI  CPV  CCV                                        cola                                         Ictero.                          ______________________________________                                        DA.sub.2 PPvL/H.sub.2 O                                                                 Neg.   724     11   74   ND   41   493                              DA.sub.2 PPv/Lepto                                                                      84     388     24   56   ND   230  800                              DA.sub.2 PPV/H.sub.2 O                                                                  128    549     7    91   ND   ND   ND                               DA.sub.2 PPVL/H.sub.2 O                                                                 223    891     9    58   ND   264  1213                             DA.sub.2 PPVL/AlOH.sub.3                                                                149    380     13   47   ND   429  2000                             DA.sub.2 PPvL/CV                                                                        91     661     7    92   6    566  1213                             ______________________________________                                         .sup.a antibodies directed against leptospira bacteria and canine viruses     CDV  canine distemper virus, CAV.sub.2  canine adenovirus type 2, CPI         canine parainfluenza virus, CPV  canine parvovirus, CCV  canine               coronavirus.                                                                  .sup.b Antibody responses are expressed as virus neutralization titers        expect for CPV and leptospira which are expressed as HI and                   microagglutination titers, respectively.                                      .sup.c Animals were vaccinated subcutaneously two times at three weeks        apart with a 1 ml dose. Serum was collected at 2 weeks after the second       vaccination.                                                                  .sup.d Indicates lyophilized vaccine components and the diluent used for      rehydration: D  distemper virus, A.sub.2  adenovirus type 2, P                parainfluenza virus, PV parvovirus, L leptospira, Cv  coronavirus.       

The results presented in Table 1 show that the coronavirus vaccine whenused to rehydrate the DA₂ PPVL vaccine, i.e., a vaccine containingdistemper virus (D), adenovirus type 2 (A₂), parainfluenza virus (P),parvovirus (PV) and leptospiral bacteria (L), did not interfere with thedevelopment of antibody to any of the viral or leptospiral components.Furthermore, no significant difference in antibody titers to any of thefractions was observed in puppies vaccinated with the DA₂ PPPVLrehydrated with the coronavirus vaccine or when water or adjuvant wasused as the diluent.

Example 3

Vaccine Evaluation In Guinea Pigs

The FECV vaccine preparation was evaluated for antigen extinctionpotency by evaluating dilutions of the standardized vaccine in alaboratory animal model. Undiluted and five-fold dilutions (1:5, 1:25,and 1:125, by volume) of vaccine were prepared in DMEM, without bovineserum. Aluminum hydroxide was added to the vaccine preparations to givea final concentration of 5%, by volume.

Four groups of six guinea pigs each, 350 g size, were each administeredtwo 1 ml doses of the vaccine preparations, intramuscularly, at aninterval of 21 days apart. The guinea pigs were bled at the time ofvaccination, at the time of second vaccination and at 14 days postsecond vaccination. Table 2 illustrates the CCV serum-virusneutralization (SVN) antibody titers of the guinea pigs at the differentbleeding times.

                  TABLE 2                                                         ______________________________________                                        Antigen Extinction Potency Evaluation in Guinea Pigs                          Serum-virus Neutralization Antibody Titers                                            Guinea             SVN Antibody Titer                                 Vaccine Pig No. Pre-Vac.   Pre-2nd Vac.                                                                           Post 2nd Vac.                             ______________________________________                                        Undiluted                                                                             1-1     Neg        8        32                                                1-2     Neg        8        32                                                1-3     Neg        8        64                                                1-4     Neg        8        32                                                1-5     Neg        8        128                                               1-6     Neg        8        256                                       1:5     2-1     Neg        Neg      8                                                 2-2     Neg        Neg      128                                               2-3     Neg        2        8                                                 2-4     Neg        2        2                                                 2-5     Neg        2        8                                                 2-6     Neg        Neg      2                                         1:25    3-1     Neg        Neg      Neg                                               3-2     Neg        Neg      Neg                                               3-3     Neg        Neg      Neg                                               3-4     Neg        Neg      Neg                                               3-5     Neg        Neg      Neg                                               3-6     Neg        Neg      Neg                                       1:125   4-1     Neg        Neg      Neg                                               4-2     Neg        Neg      Neg                                               4-3     Neg        Neg      Neg                                               4-4     Neg        Neg      Neg                                               4-5     Neg        Neg      Neg                                               4-6     Neg        Neg      Neg                                       Controls                                                                              5-1     Neg        Neg      Neg                                               5-2     Neg        Neg      Neg                                               5-3     Neg        Neg      Neg                                               5-4     Neg        Neg      Neg                                               5-5     Neg        Neg      Neg                                               5-6     Neg        Neg      Neg                                       ______________________________________                                    

All guinea pigs were SVN seronegative at the time of the firstvaccination. At the time of second vaccination the undiluted vaccineelicited an antibody response in all guinea pigs. Except for the 1:5dilution, all other dilutions did not elicit a detectable antibodyresponse at the time of second vaccination. At 14 days post secondvaccination, the geometric mean antibody titer of the guinea pigsvaccinated with the undiluted vaccine increased to 64. At this sampling,the geometric mean antibody titer of the guinea pigs vaccinated with the1:5 vaccine dilution increased to 8. In contrast, guinea pigs vaccinatedwith the other vaccine dilutions, as well as the controls, remainednegative. These data indicate that the guinea pig can serve as alaboratory model for testing FECV vaccines and that the amount ofimmunizing antigen in the vaccine dose can be diluted beyond a pointwhich stimulates an immune response These data also indicate that FECVelicits an antibody response in guinea pigs which will neutralize CCVantigens.

Example 4

Vaccine Evaluation In Puppies

The same antigenic extinction FECV vaccine preparations were evaluatedin young puppies using the gut protection test as a measure ofprotection. Young healthy puppies determined to be susceptible to CCV,were housed in isolation facilities throughout the duration of theexperimental testing. A total of 12 puppies were each vaccinated twicesubcutaneously with the vaccine preparation, at an interval of 21 daysapart. At 14 days post second vaccination, the vaccinates, along withthree nonvaccinated controls, were challenged with virulent CCV.

                  TABLE 3                                                         ______________________________________                                        Antigen Extinction Potency Evaluation in Puppies                              Serum-virus Neutralization Antibody Titers                                            Puppy   SVN Antibody Titer                                            Group   No.     Pre-Vac.   Pre-2nd Vac.                                                                           Post-2nd Vac.                             ______________________________________                                        Undiluted                                                                             64      Neg        4        32                                                74      Neg        2        32                                                82      Neg        8        64                                        1:5     66      Neg        4        32                                                76      Neg        4        16                                                84      Neg        4        32                                        1:25    68      Neg        4        8                                                 78      Neg        4        4                                                 89      Neg        4        8                                         1:125   70      Neg        Neg      4                                                 80      Neg        8        8                                                 83      Neg        Neg      Neg                                       Controls                                                                              72      Neg        Neg      Neg                                               85      Neg        Neg      Neg                                               88      Neg        Neg      Neg                                       ______________________________________                                    

At the time of vaccination, all puppies were seronegative for CCV. Atthe time of second vaccination, the undiluted, 1:5 and 1:25 vaccineselicited an immune response to CCV in all the vaccinated puppies. PuppyNo. 80 was the only puppy vaccinated with the 1:125 dilution which hadan antibody titer for CCV. At the time of challenge, all puppiesvaccinated with the undiluted, 1:5 and 1:25 dilutions of the FECVvaccine had an anamnestic immune response as indicated by the increasein CCV antibody titers. Two of the three puppies in the 1:125 vaccinatedgroup had antibody titers at the time of challenge. These data indicatethat the FECV vaccine elicits an immune response in puppies which iscapable of neutralizing CCV. Also, these data illustrate the antigenicextinction of the FECV immunogens in the vaccine.

Example 5

Challenge Experiments

For challenge, each vaccinate, along with three nonvaccinated controls,was administered one ml orally and one ml intranasally of a virulentvirus preparation. At five days post challenge, the 15 puppies wereeuthanized, necropsied and the small intestine removed. The smallintestine was divided into 10 segments of equal distance apart and asmall sample from each of the segments collected and stored at -70° C.until evaluated. For evaluation, the intestinal samples were sectioned(6 μm thick) on a cryostat, air dried on a glass slide for 30 minutes at37° C. and then stored at 4° C. until stained. For immunofluorescence,the sections were fixed in cold acetone for 10 minutes and then airdried for 30 minutes at 20° C. prior to staining with dilutedfluorescein isothiocyanate conjugated antiserum for 20 minutes at 37° C.in a humidified chamber. After staining, the slides were washed twice (5minutes each) with phosphate buffered saline (PBS), pH 7.0, and once for5 minutes with distilled water. Slides were allowed to air dry andexamined immediately by ultraviolet microscopy to determine the degree(scale of 1+ to 4+) of CCV antigens in the absorptive cells of theintestinal villi. Table 4 illustrates the results of the gut protectiontest.

                  TABLE 4                                                         ______________________________________                                        Antigen Extinction Potency Evaluation in Puppies                              Gut Protection Test                                                                 Puppy    Intestinal Segment                                             Vac.  No.      1     2   3   4    5   6   7   8   9   10                      ______________________________________                                        Undil.                                                                              64       -     -   -   +    3+  4+  2+  2+  2+  +                             74       -     -   -   +    3+  3+  3+  2+  +   +                             82       -     -   -   +    2+  +   2+  +   +   +                       1:5   66       -     -   -   -    +   2+  2+  2+  +   -                             76       -     -   -   -    +   +   +   +   +   +                             84       -     -   -   -    -   -   +   +   -   -                       1:25  68       -     -   -   -    +   -   -   -   -   -                             78       -     -   -   -    +   +   -   -   -   -                             89       -     -   -   -    -   -   -   -   -   -                       1:125 70       -     -   -   -    -   -   -   -   -   -                             80       -     -   -   -    -   -   -   -   -   -                             83       -     -   -   -    -   -   -   -   -   -                       Cntrl.                                                                              72       -     -   -   -    -   -   -   -   -   -                             85       -     -   -   -    -   -   -   -   -   -                             88       -     -   -   -    -   -   -   -   -   -                       ______________________________________                                    

These data indicate that sufficient FECV immunogens were present in theundiluted and 1:5 vaccines to prime immunocompetent cells lining themucosal membranes of the small intestine. The CCV challenge virusentered the gut and limited replication occurred. At the time ofnecropsy, at five days post-challenge, remnants of the challenge viruswere observed by the specific CCV fluorescence present in the absorptivecells lining the villi of the intestine. The 1:25 dilution of thevaccine had insufficient antigen to prime the gut as indicated by thelow amount of fluorescence observed in one and two sections from thevaccinated puppies. The 1:125 vaccine dilution failed to prime the gutand the negative fluorescence was comparable to that observed for thecontrol puppies. These data indicate that FECV immunogens can prime theimmunocompetent cells lining the intestinal mucosa and protect puppiesagainst gastroenteritis caused by CCV. The FECV immunogens in thevaccine can be diluted beyond protection and must be standardized tohave sufficient potency to protect dogs against CCV infection.

Example 6

Immunogenicity Test

A) Materials and Methods

1. Dogs

Beagle puppies were obtained from Harlan Sprague Dawley, Inc. Puppieswere six weeks old at vaccination and were seronegative (SVN antibodytiters≦2) to canine coronavirus (CCV) canine distemper virus (CDV),canine adenovirus type 2 (CAV-2), canine parvovirus (CPV) canineparainfluenza virus (CPI) and microagglutination titers of ≦10 toLeptospira canicola and L. icterohaemorrhagiae. Puppies were placed inisolation facilities throughout the duration of the vaccination andchallenge test periods.

2. Vaccines/vaccination

Virus fluids were prepared from FECV at the fifth passage from MasterSeed Virus on FK-CU cells according to the method disclosed in Example1, supra. After chemical inactivation of the virus fluids, thecoronavirus vaccine was formulated at the minimal immunizing dose ofFECV antigen. Each one ml dose contained a 1:10 dilution of inactivatedFECV fluids. The coronavirus vaccine was adjuvanted with 5% aluminumhydroxide. Experimental groups included puppies vaccinatedsubcutaneously or intramuscularly with two one ml does at three weeksapart with the coronavirus vaccine. An additional group of puppiesreceived the coronavirus vaccine in combination with a vaccinecomposition designated DA₂ PPVL which contained the minimum release doseof lyophilized modified live virus components of canine distemper virus(CDV), canine adenovirus type 2 (CAV-2), canine parainfluenza virus(CPI), canine parvovirus (CPV), and the lyophilized killed leptospirabacterin. A group of nonvaccinated puppies served as controls.

3. Serology

Blood samples for serological evaluation of SVN antibody titers werecollected before and after vaccination and after challenge with virulentCCV. Determination of SVN antibody to CCV, and CDV and CAV-2 wasperformed by a microtitration method with flat-bottom 96-well microtiterplates. Two-fold serial dilutions of serum samples were prepared inDulbecco's Modified Eagle's Medium (DMEM) maintenance medium and anequal volume (50 μl) of the appropriate virus strain containing 100-300log₁₀ and TCID₅₀ was added to each well After 90 minutes incubation at37° C., an equal volume of an appropriate cell suspension in DMEM+5% FBSadded to each well. Plates were incubated for seven days at 37° C. in ahumidified carbon dioxide incubator and examined under an invertedmicroscope for viral cytopathic effect (CPE). SVN antibody to CPI wasdetermined by the hemmadsorption inhibition technique. SVN antibody toCPV was assessed by the fluorescent antibody assay performed in 8-wellchamber slides. Antibody titers are expressed as the reciprocal of thehighest dilution of serum that completely neutralized the virus. Serumsamples were sent by the Veterinary Diagnostic Laboratory, University ofMinnesota for evaluation of antibody titers to the leptospira fractionsby the microagglutination test.

4. Challenge of puppies with virulent CCV

The CCV-6 was obtained from National Veterinary Services Laboratories(NVSL) in Ames, Iowa. A working stock of the virus containing 7.1 log₁₀TCID₅₀ /ml was prepared by passage of the virus on the certified FK-CUcell line and stored frozen at -70° C. Animals were challenged accordingto instructions of NVSL. At two weeks post second vaccination, puppieswere challenged with challenge virus diluted 1:100 in DMEM. Each puppyreceived 1 ml intranasal and 1 ml orally to give a total challengevolume of 2 ml containing a theoretical dose of 5.4 log₁₀ TCID₅₀.Titration of the diluted challenge virus resulted in an actual challengedose of 5.1 log₁₀ TCID₅₀. Fecal samples were collected daily throughoutthe 21 day post challenge period.

5. Gut protection assay

The gut-protection assay was performed essentially as described by Acreeet al. in U.S. Pat. No. 4,567,042 and U.S. Pat. No. 4,567,043. On day 21post challenge, puppies were euthanized and necropsied to harvest theintestinal tract. Ten segments, each of approximately 10 cm in lengthwere taken from the pyloric valve to the large intestine. The sectionswere placed in mounting fixative and frozen at -70° C. Thin sectionswere prepared on a microtome, air dried on glass slides and fixed andstained with fluorescein-labeled goat anti-cat IgG (H+L) antibody.Sections were examined with a UV microscope. The degree of fluorescencein each section was indicative of the amount of CCV antigen.Fluorescence intensity was scored - for negative and from ± to 4± forpositive and assigned values of 0 for negative and 0.5 to 4 forpositive. Total scores were used to calculate a protective index.

6. Isolation of CCV from feces post challenges

A 30% suspension of each fecal sample was prepared in PBS. The materialwas vortexed vigorously to break up clumps, centrifuged at 1200×g for 15minutes at 4° C. to pellet debris. The supernatant was filtered througha 0.2 μm filter and 200 μl of each sample was added to a well of 24-wellplates containing a 24-hour monolayer of feline cells. Cultures wereincubated at 37° C. in CO₂ incubator for seven days. To circumvent theproblem of toxicity associated with fecal material and increase thechances to isolate virus, 100 μl was removed from each well after fourdays incubation, and subcultured to 24-well plates containing fresh24-hour cultures of feline cells. The second set of 24-well cultureswere incubated at 37° C. in a CO₂ incubator. Both sets of cultures wereexamined for CCV-characteristic CPE after four and seven daysincubation. Cultures that exhibited CPE in the initial 24-well cultureor in the subculture were scored as positive. Positive cultures wereconfirmed as canine coronavirus by immunofluorescence. The total numberof samples tested in the initial and subculture plates was used tocalculate percentage of puppies shedding CCV in feces (% positive)

7. Detection of mucosal antibody specific for the S-protein of CCV byELISA

Wells of 96-well microtiter plates were coated with 100 μl of an optimalconcentration of a monoclonal antibody to S-protein of CCV or 100 μl ofuninfected cell control antigen for 18 hours at 4° C. Wells were washedwith PBS-0.5% Tween 20 PBS-Tw and post-coated with PBS containing 2%bovine serum albumin (PBS-BSA). After 30 minutes incubation at 37° C.,aliquots of 50 μl of CCV antigen were added to wells and incubated for 1hour at 37° C. A 50 μl aliquot of the processed and clarified fecalsample was added to wells containing CCV and cell control antigen. Afterincubation for 1 hour at 37° C., 50 μl of peroxidase-labeled goatanti-dog IgA, α-chain specific, (Bethyl Laboratories, Inc.) was added.Plates were incubated at 37° C. for 1 hour, washed and 100 μl of ABTSsubstrate (Kirkegaard & Perry Laboratories, Inc.) was added to thewells. After incubation at room temperature for 15-30 minutes,absorbance values were determined on an ELISA reader at 405 nm.Absorbance values from cell control antigen wells were subtracted fromabsorbance of CCV antigen wells and divided by the background absorbancevalue. Results were expressed as the fold-increase in antibody levels.

8. CCV SVN activity in fecal samples

Fecal samples that had been processed for virus isolation were testedfor VN activity to CCV. Prior to use in the test, the filtered fecalsample was treated with binary ethyleneimine (BEI) to inactivateresidual CCV. The BEI was neutralized by the addition of sodiumthiosulfate and the sample was tested in the CCV SVN assay

B. Results

Serum SVN antibody titers post vaccination and challenge

Immunogenicity of the coronavirus vaccine was demonstrated by thedevelopment of SVN to CCV in vaccinated puppies. Puppies vaccinated atsix weeks of age with two does of coronavirus vaccine formulated at theminimum immunizing dose developed SVN antibody to CCV (Table 5). Therewas no difference in SVN antibody titers to CCV in puppies vaccinated byeither the subcutaneous or intramuscular route. Puppies vaccinated withthe coronavirus vaccine in combination with vaccine containing a fullantigen dose of CDV, CAV-2, CPI, CPV and L. canicola and L.icterohaemorrhagiae developed SVN antibody titers to CCV that weresimilar to puppies that received the coronavirus vaccine alone. Allnonvaccinated control puppies remained seronegative to CCV throughoutthe experimental vaccination period. When puppies were challenged withvirulent CCV, a booster response CCV SVN titers was observed in allvaccinated puppies. Even the vaccinate that was seronegative after thesecond vaccination had a SVN titer similar to the other vaccinates postchallenge. CCV SVN titers in all nonvaccinated puppies did not increaseabove 8 after challenge.

                  TABLE 5                                                         ______________________________________                                        Canine Coronavirus Vaccine                                                    Killed Virus                                                                  Immunogenicity Study                                                          Serum Virus Neutralization Antibody Titers                                                    Serum SVN antibody titers                                     Dog #  Vaccine  Pre-Vac Pre-2nd Vac                                                                           Pre-Chlg.                                                                            Post Chlg..sup.1                       ______________________________________                                        839    CV (sc)  Neg.    Neg.    8      32                                     862             Neg.    Neg.    2      16                                     855             Neg.    Neg.    Neg.   16                                     868             Neg.    Neg.    4      32                                     850             Neq.    Neg.    2      64                                            GMT      Neg.    Neg.    3      29                                     856    CV (im)  Neg.    Neg.    4      32                                     857             Neg.    4       4      32                                     848             Neg.    Neg.    4      32                                     851             Neg.    Neg.    2      64                                     858             Neg.    Neg.    4      32                                            GMT      Neg.    Neg.    4      38                                     866    CV (sc)  Neg.    Neg.    8      16                                     841    with     Neg.    Neg.    8      16                                     864    DAPPvL.sup.2                                                                           Neg.    Neg.    2      16                                     847             Neg.    Neg.    8      16                                     845             Neg.    Neg.    4      32                                            GMT      Neg.    Neg.    5      19                                     854    Controls Neg.    Neg.    Neg.   8                                      849             Neg.    Neg.    Neg.   8                                      860             Neg.    Neg.    Neg.   8                                      853             Neg.    Neg.    Neg.   4                                      843             Neg.    Neg.    Neg.   4                                             GMT      Neg.    Neg.    Neg.   6                                      ______________________________________                                         .sup.1 Two weeks post CCV challenge.                                          .sup.2 Vaccine contained the full antigen release dose of CDV, CAV2, CPI,     CPV and L. canicola and L. icterohaemorragiae.                           

Gut protection assay

Puppies were challenged with virulent CCV at two weeks after the seconddose of vaccine Twenty-one days later gut segments were harvested andprocessed for evaluation in the gut protection assay. The degree offluorescence in gut sections was proportional to the amount of CCVinfection in the intestine. Protection of vaccinated puppies againstvirulent CCV was determined by a reduction in fluorescence intensityobserved in the gut sections. Efficacy of the coronavirus vaccine wasassessed by a reduction in florescence intensity of gut sections fromvaccinates compared to nonvaccinated controls. Less fluorescence wasobserved in gut sections from vaccinated puppies than in sections fromnonvaccinated control (Table 6). A reduction in fluorescence intensitywas observed in gut sections from 14 to 15 (93%) of vaccinated puppies.When fluorescence intensity values were used to calculate fluorescencescores, vaccinates had a mean score of 2.7 compared to a score of 24.3for the nonvaccinated controls. Fluorescence scores were used tocalculate a protective index of 89% for vaccinated puppies. Efficacy ofthe coronavirus vaccine was not affected by route of administration orby virus and bacterin components in the in the DA₂ PPVL vaccine.

                  TABLE 6                                                         ______________________________________                                        Canine Coronavirus Vaccine                                                    Killed Viriis                                                                 Immunogenicity Study                                                          Gut Protection Test                                                                  Fluorescence in thin sections from gut segments.sup.1                  Dog #  Vaccine  1     2   3   4   5   6   7   8   9    10                     ______________________________________                                        839    CV (sc)  -     -   -   -   -   -   -   -   -    -                      862             -     -   -   -   2+  ±                                                                              ±                                                                              ±                                                                              2+   1+                     855             -     -   -   -   -   -   -   -   -    -                      868             -     -   -   -   -   -   -   -   -    -                      850             -     3+  1+  1+  2+  1+  1+  1+  3+   2+                     856    CV (im)  -     -   -   -   -   -   -   -   -    -                      857             -     -   2+  2+  -   ±                                                                              ±                                                                              -   -    -                      848             -     ±                                                                              ±                                                                              ±                                                                              ±                                                                              ±                                                                              ±                                                                              ±                                                                              ± ±                   851             -     -   -   -   -   -   -   -   -    -                      858             -     -   -   -   -   -   -   -   -    -                      866    CV (sc)  -     -   -   ±                                                                              ±                                                                              -   ±                                                                              ±                                                                              -    ±                   841    with     -     -   -   -   -   -   -   -   -    -                      864    DA.sub.2 PPVL                                                                          -     -   -   -   -   -   -   -   -    -                      847             ±  ±                                                                              ±                                                                              ±                                                                              -   ±                                                                              +   ±                                                                              ± ±                   845             -     -   -   -   -   -   ±                                                                              ±                                                                              -    -                      854    Controls 1+    2+  4+  2+  2+  3+  -   2+  3+   3+                     849             3+    4+  3+  2+  3+  3+  4+  3+  4+   2+                     860             ±  ±                                                                              1+  3+  2+  3+  3+  2+  ± 2+                     853             4+    4+  3+  3+  1+  2+  1+  1+  2+   2+                     843             3+    3+  2+  2+  4+  3+  3+  2+  3+   3+                     ______________________________________                                         .sup.1 Degree of fluorescence scored from ± to 4+, or as  for negative     is proportional to the amount of CCV infection.                          

Isolation of CCV from feces post CCV challenge

Fecal samples collected daily throughout the 21 day post CCV challengeperiod were tested for the presence of CCV by culture on feline cells.Cultures were passaged on feline cells to amplify CCV present in thefecal sample and reduce the inherent problem of toxicity associated withfecal material. For up to three days after CCV challenge, CCV wasreisolated from approximately the same percentage of vaccinated andnonvaccinated puppies (Table 7). After three days post challenge, fewervaccinated puppies shed CCV than controls. CCV was isolated from only13% to 33% of vaccinated puppies on day 5 through 20 post challenge. Incontrast, 80% to 100% of the nonvaccinated puppies shed virus from days3 to 11 throughout the post challenge period. CCV continued to bereisolated from 40% to 100% of the puppies on days 13 through 20 postchallenge. Shedding of CCV was reduced in puppies vaccinatedsubcutaneously and intramuscularly and when the coronavirus vaccine wasused in combination with the DA₂ PPVL vaccine.

                                      TABLE 7                                     __________________________________________________________________________    Canine Coronavirus Vaccine                                                    Killed Virus                                                                  Immunogenicity Study                                                          Isolation of CCV from Feces Post Challenge                                              Days post CCV challenge                                             Dog #                                                                              Vaccine                                                                            0 1 3 5  7  9 11 13                                                                              15                                                                              17 20                                          __________________________________________________________________________    839  CV (sc)                                                                            - + - -  +  + -  - - +  +                                           862       - + + -  -  - -  - + +  +                                           855       - + - -  +  - +  - - NS.sup.1                                                                         -                                           868       - - + -  +  + -  + - -  -                                           850       - - + -  -  - +  - - -  -                                           856  CV (im)                                                                            - + + -  T.sup.2                                                                          - -  - + -  +                                           857       - - + NS -  - -  - - -  -                                           848       - - + -  -  - +  - - -  -                                           851       - - + -  -  - -  - - -  -                                           858       - - + -  -  - -  - - -  -                                           866  CV (sc)                                                                            - + + +  -  + -  - - -                                              841  with - + T +  +  + -  + - NS -                                           864  DA.sub.2 PPVL                                                                      + - T T  T  + +  - - -  -                                           847       + - T NS -  - +  + - -  -                                           845       - - + -  -  - -  - - -  -                                           % positive                                                                              13                                                                              40                                                                              67                                                                              15 27 33                                                                              33 27                                                                              13                                                                              15 20                                          854  Controls                                                                           NS                                                                              - + NS +  + +  + + +  +                                           849       - - + +  +  - NS + T +  -                                           860       - - + NS +  + NS - + +  +                                           853       + + + +  +  + NS - + +  -                                           843       - - - +  +  + +  NS                                                                              + +  -                                           % positive                                                                              25                                                                              20                                                                              80                                                                              100                                                                              100                                                                              80                                                                              100                                                                              50                                                                              80                                                                              100                                                                              40                                          __________________________________________________________________________     .sup.1 No sample.                                                             .sup.2 Toxic.                                                            

S-protein specific mucosal IgA antibody in fecal samples

To determine levels of mucosal antibody to CCV, fecal samples weretested in an ELISA that measures IgA antibody specific for the S-proteinof CCV. IgA levels for individual vaccinated and control puppies arepresented in Table 8 and a summary of IgA levels is presented in FIG. 1.At five days post CCV challenge, a 4 to 13 fold increase in S-proteinspecific IgA was detected in fecal samples from vaccinated puppies. Atthis same time, nonvaccinated control puppies showed a maximum of a twofold increase in IgA. Levels of S-protein specific IgA remained elevatedin vaccinated animals through day 13 post challenge. IgA levelsgradually decreased in vaccinated puppies after day 14 but remainedhigher than control puppies. Although IgA levels in some nonvaccinatedcontrol puppies increased as much as five fold, the mean IgA levels inthese animals remained low. There was little difference in IgA levelsamong the puppies vaccinated subcutaneously and intramuscularly with thecoronavirus vaccine alone and in combination with the DA₂ PPVL vaccine.

                                      TABLE 8                                     __________________________________________________________________________    Canine Coronavirus Vaccine                                                    Killed Virus                                                                  Immunogenicity Study                                                          S-Protein Specific Mucosal IgA Antibody                                                Fold increase in S-protein                                                    specific IgA on days post CCV challenge                              Dog #                                                                             Vaccine                                                                            0 1 3 5  7  9  11 13 15                                                                              17 20                                         __________________________________________________________________________    839 CV (sc)                                                                            1.0                                                                             1.0                                                                             1.0                                                                             NS 8.9                                                                              10.2                                                                             10.8                                                                             11.5                                                                             4.9                                                                             3.3                                                                              4.6                                        862      1.0                                                                             1.0                                                                             1.0                                                                             4.2                                                                              11.3                                                                             11.3                                                                             9.6                                                                              7.5                                                                              7.0                                                                             5.6                                                                              3.2                                        855      1.0                                                                             1.3                                                                             1.0                                                                             8.9                                                                              9.1                                                                              7.9                                                                              10.4                                                                             9.1                                                                              4.3                                                                             NS 2.8                                        868      1.0                                                                             1.0                                                                             1.0                                                                             13.1                                                                             11.9                                                                             8.9                                                                              10.4                                                                             5.8                                                                              4.9                                                                             6.5                                                                              3.5                                        850      1.0                                                                             1.0                                                                             1.0                                                                             6.9                                                                              9.2                                                                              14.6                                                                             10.4                                                                             8.8                                                                              3.1                                                                             4.8                                                                              3.9                                        856 CV (im)                                                                            1.9                                                                             1.0                                                                             1.0                                                                             8.5                                                                              5.8                                                                              9.0                                                                              8.0                                                                              8.4                                                                              2.7                                                                             3.1                                                                              1.8                                        857      1.0                                                                             1.0                                                                             1.0                                                                             NS 10.7                                                                             8.2                                                                              10.5                                                                             11.2                                                                             9.2                                                                             2.9                                                                              2.2                                        848      1.0                                                                             1.0                                                                             1.0                                                                             NS 6.6                                                                              5.4                                                                              6.7                                                                              7.8                                                                              2.4                                                                             5.0                                                                              1.7                                        851      1.0                                                                             1.0                                                                             2.3                                                                             6.1                                                                              4.5                                                                              3.3                                                                              5.3                                                                              6.0                                                                              4.1                                                                             5.1                                                                              2.2                                        858      1.0                                                                             1.0                                                                             1.0                                                                             6.2                                                                              8.3                                                                              6.4                                                                              5.1                                                                              6.6                                                                              4.1                                                                             6.6                                                                              3.5                                        866 CV (sc)                                                                            1.0                                                                             1.0                                                                             2.7                                                                             6.0                                                                              5.3                                                                              5.2                                                                              4.8                                                                              2.5                                                                              7.9                                                                             NS 6.0                                        841 with 1.0                                                                             1.0                                                                             1.0                                                                             4.9                                                                              8.7                                                                              4.9                                                                              8.3                                                                              6.5                                                                              4.4                                                                             NS 3.6                                        864 DA.sub.2 PPVL                                                                      1.0                                                                             1.0                                                                             1.0                                                                             6.5                                                                              6.1                                                                              3.4                                                                              7.4                                                                              9.6                                                                              5.4                                                                             7.4                                                                              3.1                                        847      1.8                                                                             1.0                                                                             1.0                                                                             NS 5.3                                                                              8.2                                                                              5.8                                                                              7.4                                                                              3.8                                                                             7.9                                                                              4.7                                        845      1.0                                                                             1.0                                                                             1.0                                                                             8.4                                                                              6.3                                                                              7.1                                                                              6.1                                                                              5.7                                                                              4.3                                                                             4.9                                                                              4.8                                        854 Controls                                                                           NS                                                                              1.0                                                                             1.0                                                                             NS NS 3.1                                                                              2.4                                                                              3.5                                                                              3.1                                                                             1.9                                                                              1.1                                        849      1.1                                                                             1.0                                                                             1.8                                                                             1.7                                                                              1.6                                                                              2.2                                                                              NS 1.5                                                                              2.2                                                                             1.9                                                                              2.2                                        860      1.8                                                                             1.1                                                                             2.3                                                                             NS 1.1                                                                              1.0                                                                              NS 1.0                                                                              1.3                                                                             1.0                                                                              4.6                                        853      1.0                                                                             1.0                                                                             1.0                                                                             1.4                                                                              1.3                                                                              1.2                                                                              1.1                                                                              5.1                                                                              1.0                                                                             1.3                                                                              3.1                                        843      2.0                                                                             1.0                                                                             1.0                                                                             2.1                                                                              1.0                                                                              1.6                                                                              NS NS 1.0                                                                             1.0                                                                              1.3                                        __________________________________________________________________________

CCV SVN activity in mucosal samples

Fecal samples were also tested to determine the presence of mucosalCCV-neutralizing activity. Since CCV present in the filtered fecalsample may interfere with the CCV used for the SVN assay, fecal sampleswere treated with BEI to inactivate virus. The sample was then testedfor the presence of SVN activity to CCV as described for the CCV SVNantibody assay. Mucosal CCN SVN titers of ≧64 were detected in the fecesof several vaccinated puppies and persisted for more than one day (Table9). Mucosal SVN titers were similar in fecal samples from all threevaccinate groups. As with the mucosal antibody ELISA, mucosal CCV SVNactivity was detected in fecal samples from nonvaccinated puppies postCCV challenge. However, mucosal SVN titers in controls were generallylower and varied more day to day and from animal to animal

                                      TABLE 9                                     __________________________________________________________________________    Canine Coronavirus Vaccine                                                    Killed Virus                                                                  Immunogenicity Study                                                          Mucosal CCV SVN Activity in Fecal Samples                                              Days post CCV challenge                                              Dog #                                                                             Vaccine                                                                            0 1  3  5 7  9  11                                                                              13                                                                              15 17 20                                         __________________________________________________________________________    839 CV (sc)                                                                            8 8  8  16                                                                              32 ≧64                                                                       16                                                                              32                                                                              32 ≧64                                                                       32                                         862      16                                                                              16 16 NS                                                                              16 32 16                                                                              16                                                                              8  16 16                                         855      16                                                                              16 16 32                                                                              ≧64                                                                       8  16                                                                              16                                                                              32 32 T                                          868      T 8  8  32                                                                              8  ≧64                                                                       32                                                                              8 32 32 32                                         850      8 16 8  16                                                                              32 16 8 16                                                                              32 8  32                                             GMT  11                                                                              12 11 23                                                                              24 28 16                                                                              16                                                                              24 24 27                                         856 CV (im)                                                                            8 8  8  8 T  32 16                                                                              16                                                                              32 8  8                                          857      8 8  T  NS                                                                              16 32 32                                                                              32                                                                              32 8  ≧64                                 848      8 8  16 32                                                                              16 32 32                                                                              32                                                                              32 8  32                                         851      16                                                                              16 32 8 32 16 16                                                                              16                                                                              16 16 T                                          858      8 16 32 16                                                                              16 32 16                                                                              16                                                                              8  16 8                                              GMT  9 11 19 14                                                                              19 28 21                                                                              21                                                                              21 11 19                                         866 CV (sc)                                                                            16                                                                              ≧64                                                                       ≧64                                                                       16                                                                              32 32 16                                                                              16                                                                              32 16 32                                         841 with 16                                                                              8  32 8 16 16 8 32                                                                              32 ≦8                                                                        16                                         864 DA.sub.2 PPVL                                                                      32                                                                              16 16 32                                                                              16 16 16                                                                              32                                                                              ≧64                                                                       8  32                                         847      16                                                                              32 ≧64                                                                       8 16 16 32                                                                              16                                                                              8  8  8                                          845      16                                                                              16 16 32                                                                              8  8  16                                                                              8 16 16 16                                             GMT  18                                                                              21 32 16                                                                              16 16 16                                                                              16                                                                              24 7  18                                         854 Controls                                                                           NS                                                                              8  8  NS                                                                              8  8  8 ≦8                                                                       8  8  16                                         849      8 16 32 16                                                                              16 8  ≦8                                                                       8 8  8  T                                          860      32                                                                              ≦8                                                                        32 NS                                                                              16 16 ≦8                                                                       ≦8                                                                       ≦8                                                                        ≦8                                                                        8                                          853      8 16 16 32                                                                              T  T  8 8 8  32 ≦8                                  843      8 8  16 16                                                                              8  32 ≦8                                                                       ≦8                                                                       8  8  ≦8                                  843 GMT  11                                                                              7  18 20                                                                              11 14 2 2 5  7  4                                          __________________________________________________________________________

Discussion

CCV enteritis continues to be an important disease of the dog. Althoughthe course of the disease can be mild, factors such as stress, breed andother canine pathogens can enhance the severity of the disease (3).Thus, CCV vaccines are needed to maintain the health of the dog.

A strain of FECV was chosen as a candidate for further vaccinedevelopment because of the growth characteristics and antigenicity ofthe virus. These characteristics facilitated the economic development ofan efficacious vaccine prepared from inactivated and adjuvanted FECVfluids. Immunogenicity of the coronavirus vaccine was demonstrated inpuppies as young as six weeks of age by the development of CCV SVNantibody. Results showing that similar SVN antibody titers observed inpuppies vaccinated by the subcutaneous or intramuscular route indicatedthe vaccine can be administered by either route. In addition,combination of the coronavirus vaccine with the DA₂ PPVL vaccine did notinterfere with development of CCV VN antibody. Therefore, thecoronavirus vaccine can be used with the routine canine vaccinationregimen of virus vaccines and bacterin components.

Although severe clinical disease can be observed in unprotected dogsafter natural exposure to virulent field CCV, the CCV challenge virusprovided by NVSL does not produce clinical symptoms in normal puppies.Therefore, efficacy of the coronavirus vaccine was evaluated by the gutprotection assay described by Acree et al. in U.S. Pat. No. 4,567,042and U.S. Pat. No. 4,567,043. Fluorescence intensity of gut sections wasused as an indicator of a protective response after virulent CCVchallenge. The degree of fluorescence observed in the gut sections, wasproportional to the level of CCV infection present in the gut segment.Thus, a reduction in fluorescence intensity of gut sections fromvaccinated puppies was interpreted as a reduction in virus infectionpost challenge with virulent CCV. Reduced fluorescence was observed insections from 14 of 15 (93%) of vaccinated puppies compared tononvaccinated controls. These results demonstrated the protectiveability of the coronavirus vaccine to reduce viral infection afterchallenge with virulent CCV. An overall protective index of 89% wasdemonstrated for puppies vaccinated with the coronavirus vaccine aloneor in combination with the DA₂ PPVL vaccine.

Results from the gut-protection assay indicated reduced CCV infection invaccinates at the end of the challenge period. However, whetherclearance of virulent CCV in vaccinates occurred gradually or at adefinitive time point after challenge was not known. Results of thevirus isolation study showed that the decrease in the number ofvaccinates shedding CCV occurred between three and five days post CCVchallenge. The enhanced clearance of virulent CCV from vaccinatedpuppies persisted throughout the 21 day challenge period. Althoughnonvaccinated puppies did not exhibit any severe clinical symptoms postchallenge, CCV was shed from the majority of nonvaccinated puppiesduring the entire post challenge period. The shed of virus fromnonvaccinated puppies for extended periods of time such as this couldpredispose the puppy to infection by other more severe canine pathogens.

The mucosal immune response plays an important role in protectionagainst disease caused by enteric viruses. Investigators have reportedthat stimulation of the gut-associated lymphoid tissue and production ofmucosal IgA antibody is the major protective response against TGE virusinfection (14, 15, 16). Since CCV infection is not systemic, the mucosalimmune response is the predominant mechanism for clearance of CCV fromthe intestine. Because the S-protein of coronaviruses is major target ofSVN antibody, an ELISA was developed that measured levels of mucosal IgAspecific for the S-protein. Results of the ELISA demonstrated that invaccinated puppies, levels of specific mucosal IgA increased more thanseven-fold over background by day five post CCV challenge. In vaccinatedpuppies the amount of mucosal IgA correlated with the protective immuneresponse of mucosal antibody. Mucosal IgA antibody to the S-protein ofCCV was detected in nonvaccinated puppies. However, the mucosal immuneresponse in the puppies was not sufficient to reduce shedding of CCVthroughout the 21 day post challenge period. Additional evidence as tothe protective role of the mucosal immune response was provided byresults that showed the presence of CCV SVN activity in the fecalsamples. Although the CCV SVN titers in fecal samples varied, in generalVN titers were higher and persisted longer post CCV challenge invaccinates than controls.

The results presented here demonstrate the development of a safe andefficacious coronavirus vaccine. The coronavirus vaccine was efficaciousin six week old puppies when administered subcutaneously andintramuscularly and in combination with a vaccine containing thestandard virus and bacterin antigen components. Protection of vaccinatedpuppies against virulent CCV challenge was assessed by thegut-protection assay. Efficacy was demonstrated by reduced CCV infectionof gut segments from vaccinated puppies. In vaccinated puppies, thereduction in virus infection in the intestine correlated with reducedvirus shedding and elevated levels of S-protein specific IgA.

References

1. Binn et al, Proc. 78th Ann. Mtg., U.S. Anim. Health Assoc. RoanokeVa. October: 359-366 (1974).

2. Kennan et al, Am. J. Vet. Res. 37:247-256 (1976).

3. Everman et al, Comp. Anim. Pract. 2:15-23 (1988).

4. Appel et al., Canine Prac. 7:22-36 (1980).

5. Fulker et al., Canine Prac. 13:18-27 (1986).

6. Martin, Comp. Cont. Educ. Prac. Vet. 7:1012-1017 (1985).

7. Pedersen et al., Arch. Virol. 58:45-53 (1978).

8. Pensaert et al., Arch. Virol. 68:45-52 (1981).

9. Bordt et al., European Patent No. 0 310 316.

10. Reynolds et al., Arch. Virol. 60:161-166 (1979).

11. Woods et al. Vet. Microbiol. 4:11-16 (1979).

12. Oi., V. T., and L. A. Herzenberg, Immunoglobulin-producing hybridcell lines. In: Selected Methods in Cellular Immunology; (Freeman, SanFrancisco, 1980; B. B. Mishell and S. Shiigi, eds.) pp. 351-372.

13. Harlow, E. and Lane, D., Immunizations, In: Antibodies, A LaboratoryManual. (Cold Spring Harbor Laboratory 1988) pp. 53-137.

14. Aynaud, J. M. et al., Vet. Microbiol. 26:227-239 (1991).

15. Saif, L. J. et al., In: Immunology of Breast Milk P. L. Ogra and D.H. Dayton, eds. (New York, Raven Press: 1979) pp. 237-255.

16. Saif, L. J., et al. Am. J. Vet. Res. 40:115-117 (1979).

What is claimed is:
 1. A method of immunizing a dog against disease caused by canine coronavirus which comprises administering to the dog a dose of a feline enteric coronavirus vaccine comprising an effective immunizing amount of an inactivated feline enteric coronavirus and a suitable carrier.
 2. The method of claim 1, wherein the dog is at least six weeks old.
 3. The method of claim 1, wherein the dog is from about six weeks old to about nine weeks old.
 4. The method of claim 1, further comprising administering to the dog one or more additional doses of vaccine, each additional dose of vaccine being administered at a suitable interval of time after administration of the preceding dose.
 5. The method of claim 4, wherein the suitable interval of time is an amount of time from about two weeks to about five weeks.
 6. The method of claim 5, wherein the suitable interval of time is an amount of time from about two weeks to about three weeks.
 7. A method of immunizing a dog against disease caused by canine coronavirus and disease caused by a second virus which comprises administering to the dog a dose of a vaccine comprising an effective immunizing amount of an inactivated feline enteric coronavirus, an effective immunizing amount of a second inactivated virus, and a suitable carrier.
 8. A method of immunizing a dog against disease caused by canine coronavirus and disease caused by a second virus which comprises administering to the dog a dose of a vaccine comprising an effective immunizing amount of an inactivated feline enteric coronavirus, an effective immunizing amount of an inactivated bacteria, and a suitable carrier.
 9. The method of claim 1, wherein the effective immunizing amount of inactivated feline enteric coronavirus is greater than 5 micrograms of S viral antigen.
 10. The method of claim 9, wherein the effective immunizing amount of inactivated feline enteric coronavirus is an amount from about 5 micrograms to about 7.5 micrograms of S viral antigen.
 11. The method of claim 1, wherein the inactivated feline enteric coronavirus has been inactivated by contacting the virus with an agent selected from the group consisting of binary ethylenimine, formalin and β-propriolactone.
 12. The method of claim 11, wherein the inactivated feline enteric coronavirus has been inactivated by contacting the virus with binary ethylenimine.
 13. The method of claim 1, wherein the vaccine further comprises an adjuvant in an amount effective to enhance the immunogenicity of the inactivated feline enteric coronavirus.
 14. The method of claim 13, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, saponin, and aluminum phosphate.
 15. The method of claim 14, wherein the adjuvant is aluminum hydroxide.
 16. The method of claim 13, wherein the effective amount of the adjuvant is from about 3% by volume of the dose to about 10% by volume of the dose.
 17. The method of claim 13, wherein the effective amount of the adjuvant is about 5% by volume of the dose.
 18. The method of claim 7, wherein the second inactivated virus is selected from the group consisting of inactivated canine distemper virus, inactivated canine adenovirus, inactivated canine parvovirus, inactivated canine parainfluenza virus, and inactivated canine herpesvirus.
 19. The method of claim 8, wherein the inactivated bacteria is inactivated Leptospira. 